RESUMEN
VPS13 proteins are evolutionarily conserved. Mutations in the four human genes (VPS13A-D) encoding VPS13A-D proteins are linked to developmental or neurodegenerative diseases. The relationship between the specific localization of individual VPS13 proteins, their molecular functions, and the pathology of these diseases is unknown. Here we used a yeast model to establish the determinants of Vps13's interaction with the membranes of Golgi apparatus. We analyzed the different phenotypes of the arf1-3 arf2Δ vps13∆ strain, with reduced activity of the Arf1 GTPase, the master regulator of Golgi function and entirely devoid of Vps13. Our analysis led us to propose that Vps13 and Arf1 proteins cooperate at the Golgi apparatus. We showed that Vps13 binds to the Arf1 GTPase through its C-terminal Pleckstrin homology (PH)-like domain. This domain also interacts with phosphoinositol 4,5-bisphosphate as it was bound to liposomes enriched with this lipid. The homologous domain of VPS13A exhibited the same behavior. Furthermore, a fusion of the PH-like domain of Vps13 to green fluorescent protein was localized to Golgi structures in an Arf1-dependent manner. These results suggest that the PH-like domains and Arf1 are determinants of the localization of VPS13 proteins to the Golgi apparatus in yeast and humans.
Asunto(s)
Factor 1 de Ribosilacion-ADP/metabolismo , Aparato de Golgi/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Factor 1 de Ribosilacion-ADP/genética , Clatrina/metabolismo , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Microscopía Confocal , Mitocondrias/genética , Mitocondrias/metabolismo , Modelos Biológicos , Mutación , Enfermedades Neurodegenerativas/genética , Enfermedades Neurodegenerativas/metabolismo , Unión Proteica , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , TemperaturaRESUMEN
Charcot-Marie-Tooth disease (CMT) is a heritable neurodegenerative disease that displays great genetic heterogeneity. The genes and mutations that underlie this heterogeneity have been extensively characterized by molecular genetics. However, the molecular pathogenesis of the vast majority of CMT subtypes remains terra incognita. Any attempts to perform experimental therapy for CMT disease are limited by a lack of understanding of the pathogenesis at a molecular level. In this study, we aim to identify the molecular pathways that are disturbed by mutations in the gene encoding GDAP1 using both yeast and human cell, based models of CMT-GDAP1 disease. We found that some mutations in GDAP1 led to a reduced expression of the GDAP1 protein and resulted in a selective disruption of the Golgi apparatus. These structural alterations are accompanied by functional disturbances within the Golgi. We screened over 1500 drugs that are available on the market using our yeast-based CMT-GDAP1 model. Drugs were identified that had both positive and negative effects on cell phenotypes. To the best of our knowledge, this study is the first report of the Golgi apparatus playing a role in the pathology of CMT disorders. The drugs we identified, using our yeast-based CMT-GDAP1 model, may be further used in translational research.
Asunto(s)
Enfermedad de Charcot-Marie-Tooth/genética , Aparato de Golgi/genética , Proteínas del Tejido Nervioso/genética , Red trans-Golgi/genética , Enfermedad de Charcot-Marie-Tooth/patología , Heterogeneidad Genética , Aparato de Golgi/patología , Células HeLa , Humanos , Modelos Genéticos , Mutación/genética , Linaje , Relación Estructura-Actividad , Levaduras/genéticaRESUMEN
Chorein is a protein of the Vps13 family, and defects in this protein cause the rare neurodegenerative disorder chorea-acanthocytosis (ChAc). Chorein is involved in the actin cytoskeleton organization, calcium ion flux, neuronal cell excitability, exocytosis and autophagy. The function of this protein is poorly understood, and obtaining this knowledge is a key to finding a cure for ChAc. Chorein, as well as the Vps13 protein from yeast, contains the APT1 domain. Our previous research has shown that the APT1 domain from yeast Vps13 (yAPT1v) binds phosphatidylinositol 3-phosphate (PI3P) in vitro. In this study, we showed that although the APT1 domain from chorein (hAPT1) binds to PI3P it could not functionally replace yAPT1v. The hAPT1 domain binds, in addition to PI3P, to phosphatidylinositol 5-phosphate (PI5P). The binding of hAPT1 to PI3P, unlike the binding of yAPT1v to PI3P, is regulated by the bivalent ions, calcium and magnesium. Regulation of PI3P binding via calcium is also observed for the APT1 domain of yeast autophagy protein Atg2. The substitution I2771R, found in chorein of patient suffering from ChAc, reduces the binding of the hAPT1 domain to PI3P and PI5P. These results suggest that the ability of APT1 domains to bind phosphoinositides is regulated differently in yeast and human protein and that this regulation is important for chorein function.
Asunto(s)
Neuroacantocitosis/genética , Proteínas de Saccharomyces cerevisiae/genética , Tioléster Hidrolasas/genética , Proteínas de Transporte Vesicular/genética , Autofagia/genética , Proteínas Relacionadas con la Autofagia/genética , Calcio/química , Humanos , Iones/química , Magnesio/química , Mutación/genética , Neuroacantocitosis/metabolismo , Neuroacantocitosis/patología , Neuronas/metabolismo , Neuronas/patología , Fosfatos de Fosfatidilinositol/genética , Unión Proteica/genética , Dominios Proteicos/genética , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae/química , Tioléster Hidrolasas/química , Proteínas de Transporte Vesicular/químicaRESUMEN
The essential role of dolichyl phosphate (DolP) as a carbohydrate carrier during protein N-glycosylation is well established. The cellular pool of DolP is derived from de novo synthesis in the dolichol branch of the mevalonate pathway and from recycling of DolPP after each cycle of N-glycosylation, when the oligosaccharide is transferred from the lipid carrier to the protein and DolPP is released and then dephosphorylated. In Saccharomyces cerevisiae, the dephosphorylation of DolPP is known to be catalyzed by the Cwh8p protein. To establish the role of the Cwh8p orthologue in another distantly related yeast species, Candida albicans, we studied its mutant devoid of the CaCWH8 gene. A double Cacwh8∆/Cacwh8∆ strain was constructed by the URA-blaster method. As in S. cerevisiae, the mutant was impaired in DolPP recycling. This defect, however, was accompanied by an elevation of cis-prenyltransferase activity and higher de novo production of dolichols. Despite these compensatory changes, protein glycosylation, cell wall integrity, filamentous growth, and biofilm formation were impaired in the mutant. These results suggest that the defects are not due to the lack of DolP for the protein N-glycosylation but rather that the activity of oligosacharyltransferase could be inhibited by the excess DolPP accumulating in the mutant.
Asunto(s)
Candida albicans/metabolismo , Dolicoles/biosíntesis , Proteínas Fúngicas/genética , Oligosacáridos de Poliisoprenil Fosfato/metabolismo , Procesamiento Proteico-Postraduccional , Pirofosfatasas/genética , Candida albicans/crecimiento & desarrollo , Pared Celular/metabolismo , Dolicoles/genética , Proteínas Fúngicas/metabolismo , Glicosilación , Morfogénesis , Pirofosfatasas/metabolismoRESUMEN
Chorea-acanthocytosis (ChAc) is a rare neurodegenerative disease associated with mutations in the human VPS13A gene. The mechanism of ChAc pathogenesis is unclear. A simple yeast model was used to investigate the function of the single yeast VSP13 orthologue, Vps13. Vps13, like human VPS13A, is involved in vesicular protein transport, actin cytoskeleton organisation and phospholipid metabolism. A newly identified phenotype of the vps13Δ mutant, sodium dodecyl sulphate (SDS) hypersensitivity, was used to screen a yeast genomic library for multicopy suppressors. A fragment of the MYO3 gene, encoding Myo3-N (the N-terminal part of myosin, a protein involved in the actin cytoskeleton and in endocytosis), was isolated. Myo3-N protein contains a motor head domain and a linker. The linker contains IQ motifs that mediate the binding of calmodulin, a negative regulator of myosin function. Amino acid substitutions that disrupt the interaction of Myo3-N with calmodulin resulted in the loss of vps13Δ suppression. Production of Myo3-N downregulated the activity of calcineurin, a protein phosphatase regulated by calmodulin, and alleviated some defects in early endocytosis events. Importantly, ethylene glycol tetraacetic acid (EGTA), which sequesters calcium and thus downregulates calmodulin and calcineurin, was a potent suppressor of vps13Δ. We propose that Myo3-N acts by sequestering calmodulin, downregulating calcineurin and increasing activity of Myo3, which is involved in endocytosis and, together with Osh2/3 proteins, functions in endoplasmic reticulum-plasma membrane contact sites. These results show that defects associated with vps13Δ could be overcome, and point to a functional connection between Vps13 and calcium signalling as a possible target for chemical intervention in ChAc. Yeast ChAc models may uncover the underlying pathological mechanisms, and may also serve as a platform for drug testing.This article has an associated First Person interview with the first author of the paper.
Asunto(s)
Señalización del Calcio , Calcio/metabolismo , Calmodulina/metabolismo , Modelos Biológicos , Miosinas/metabolismo , Neuroacantocitosis/tratamiento farmacológico , Neuroacantocitosis/metabolismo , Saccharomyces cerevisiae/metabolismo , Citoesqueleto de Actina/efectos de los fármacos , Citoesqueleto de Actina/metabolismo , Alelos , Sustitución de Aminoácidos , Calcineurina/metabolismo , Señalización del Calcio/efectos de los fármacos , Canavanina/farmacología , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Endocitosis/efectos de los fármacos , Genes Supresores , Mutación/genética , Dominios Proteicos , Saccharomyces cerevisiae/crecimiento & desarrollo , Proteínas de Saccharomyces cerevisiae/metabolismo , Dodecil Sulfato de Sodio , Transcripción Genética/efectos de los fármacos , Vacuolas/metabolismoRESUMEN
The Vps (vacuolar protein sorting) group of proteins was identified in yeast Saccharomyces cerevisiae. Among the Vps proteins, there is the Vps13 family, proteins of which are present in organisms from different systematic groups. In yeast there is only one Vps13 protein, while in humans there are four Vps13 family members - hVps13A-D. These are large proteins of characteristic domain structure. Mutations in hVPS13 genes are linked to rare neurodegenerative disorders: chorea- acanthocytosis (hVPS13A), Cohen syndrome (hVPS13B), predispose to early onset into Parkinson disease (hVPS13C) and lead to ataxia/spastic paraplegia (hVPS13D). Lack of clear diagnostic criteria and broad spectrum of nonspecific symptoms cause the misdiagnosis of several patients with neurodegeneration and it is difficult to estimate the number of individuals with mutations in hVPS13 genes. The importance of Vps13 family proteins for human health turns interest of research on finding Vps13 protein function, which remains unknown. The research is mostly performed on several experimental models in which deficit of those proteins was acquired by deletions or gene expression silencing, or on cells from patients. Several changes were found in cells lacking Vps13 proteins on cellular level, such as changes in intracellular protein trafficking between Golgi apparatus, plasma membrane and endosomes, changes in mitochondria functioning and changes in organization of cytoskeletons, mainly actin cytoskeleton. However, it is unknown which alterations are primary and which secondary, compensatory. Recently research done on yeast revealed that Vps13 is a protein of the membrane contact sites, the structures involved in exchange of metabolites between different organelles. Such localization seems to be essential for Vps13 function. Based on literature we propose a hypothesis that Vps13 might actively participate in exchange of the lipids between membranes of organelles in membrane contact sites what could explain most of the phenotypes caused by lack of Vps13 protein.